We have recently discovered and characterized a novel hormone, intermedin/adrenomedullin 2, belonging to the CGRP/adrenomedullin peptide family. Functional studies showed that intermedin signals through a G protein-coupled receptor, calcitonin receptor-like receptor (CLR). CLR is known to mediate the action of CGRP and adrenomedullin, but efficient signaling by these hormones requires one of three coreceptors known as receptor activity-modifying proteins (RAMP1, 2, and 3). CGRP preferentially signals through cells expressing RAMP1 and CLR whereas adrenomedullin exhibits greater potency on cells expressing RAMP2 and CLR, or RAMPS and CLR. In contrast, the newly identified intermedin preferentially activates RAMP1- and RAMP-3 mediated CLR signaling. Therefore, intermedin could regulate novel physiological processes or those previously shown to be regulated by CGRP or adrenomedullin. Furthermore, we have demonstrated that intermedin exhibits potent cardiovascular effects and represents an estrogen-dependent prolactin- releasing hormone in the pituitary. Our preliminary data shows that, 1) a tethered CGRP-RAMP1 chimera constitutively activates CLR in the absence of a ligand, and 2) direct interaction of the tethered CGRP- RAMP1 chimera with CLR results in the formation of a stable complex essential for G protein coupling. Thus, we hypothesize that activation of CLR signaling by CGRP family peptides could involve three sequential steps that lead to the formation of a trimeric complex consisting of the ligand, RAMP, and CLR. Because selective interactions between CGRP family peptides with RAMP proteins are crucial in the selective activation of CLR, in Specific Aim 1 we propose to study the structural-functional characteristics of the CGRP family peptides and generate RAMP-selective agonists and antagonists. In Specific Aim 2, we will investigate the role of ligand-RAMP interactions in the formation of a trimeric ligand-RAMP-CLR complex for signaling using tethered ligand-RAMP fusion proteins. In Specific Aim 3, taking advantage of findings that the tethered CGRP-RAMP1 chimera constitutively activates CLR, we will generate and characterize transgenic mice with the tethered CGRP-RAMP1 chimera using a knock-in strategy. Because the CGRP-RAMP1 transgene is flanked by LoxP sites, we also can generate conditional RAMP1-deficient mice for future studies. Altogether, this proposal aims to answer two of the critical questions in the study of CGRP family peptides: how receptor specificity is determined and what are the physiological roles of individual RAMPs in vivo. By dissecting the molecular mechanisms underling ligand-receptor interactions and specifically investigating the RAMP1- mediated CLR signaling in transgenic mice, these experiments could facilitate the determination of functional motifs in both ligands and RAMPs for CLR activation as well as illustrate the physiological significance of individual RAMP proteins in diverse physiological processes and cardiovascular diseases.